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Research ArticleArticle

Homogeneous, Real-Time NanoBRET Binding Assays for the Histamine H3 and H4 Receptors on Living Cells

Tamara A. M. Mocking, Eléonore W. E. Verweij, Henry F. Vischer and Rob Leurs
Molecular Pharmacology December 2018, 94 (6) 1371-1381; DOI: https://doi.org/10.1124/mol.118.113373

Abstract

Receptor-binding affinity and ligand-receptor residence time are key parameters for the selection of drug candidates and are routinely determined using radioligand competition-binding assays. Recently, a novel bioluminescence resonance energy transfer (BRET) method utilizing a NanoLuc-fused receptor was introduced to detect fluorescent ligand binding. Moreover, this NanoBRET method gives the opportunity to follow fluorescent ligand binding on intact cells in real time, and therefore, results might better reflect in vivo conditions as compared with the routinely used cell homogenates or purified membrane fractions. In this study, a real-time NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the histamine H3 receptor (H3R) and histamine H4 receptor on intact cells. Obtained residence times of clinically tested H3R antagonists were reflected by their duration of H3R antagonism in a functional receptor recovery assay.

Introduction

Blockbuster antihistamines targeting the histamine H1 and H2 receptors have been used for many decades to alleviate allergy symptoms and gastric acid–related disorders, respectively (Panula et al., 2015). In contrast, drug discovery programs on the other two members of the histamine receptor family are still ongoing. The histamine H3 receptor (H3R) is predominantly expressed in the central nervous system and considered a therapeutic target for neurologic disorders, such as Alzheimer’s and Parkinson’s diseases, attention deficit hyperactivity disorder, epilepsy, and narcolepsy (Wijtmans et al., 2007; Sadek et al., 2016). The histamine H4 receptor (H4R) is predominantly expressed on immune cells and linked to inflammatory disorders, such as psoriasis, atopic dermatitis, asthma, and arthritis (Novak et al., 2012; Verweij et al., 2017). In 2016, the first H3R-targeting therapeutic, pitolisant (Wakix, Bioprojet Pharma, Paris, France), was approved by the European Medicine Agency for the treatment of narcolepsy (Kollb-Sielecka et al., 2017), whereas H4R-targeting drug candidates are in clinical trials for the treatment of various inflammatory disorders (Thurmond, 2015; Attali et al., 2016; Thurmond et al., 2017).

Traditionally, radioligands are used in competition binding experiments to indirectly determine the binding affinities (Ki), association (kon), and dissociation (koff) rate constants of the binding of unlabeled ligands to a receptor. However, these radioligand-based methods generally require physical separation of free and bound radioligands to quantify receptor-bound radioligand. Consequently, these endpoint assays are particularly labor intensive, as each measured time point requires a separate binding reaction. Recently, a novel proximity-based method was introduced for the histamine H1 receptor and a number of other G protein–coupled receptors (GPCRs) by genetic fusion of an engineered enzyme, NanoLuc (Nluc), to the N-terminus of these receptors, allowing only the detection of bound fluorescent ligand via bioluminescence resonance energy transfer (nanoBRET). Consequently, this novel method does not require physical separation of free from bound fluorescent ligand (Stoddart et al., 2015; Soave et al., 2016). Hence, this proximity-based (<10 nm) method allows real-time measurement of ligand binding to GPCRs on intact cells. Moreover, no additional prelabeling steps are required in contrast to the time-resolved fluorescence resonance energy transfer–based Tag-lite ligand-receptor-binding assay, which uses Tb3+-cryptate conjugated to an N-terminal SNAP-tagged receptor as a resonance energy transfer donor (Zwier et al., 2010; Sykes et al., 2017). The development and commercial availability of fluorescent H3R and H4R ligands urged us to set up and validate a NanoBRET-based binding assay for the H3R and H4R (Tomasch et al., 2012; Vernall et al., 2014; Mirzahosseini et al., 2015). In this study, a NanoBRET-based binding assay was established and validated to detect binding of unlabeled ligands to the human H3R (hH3R) and human H4R (hH4R) on intact cells.

Materials and Methods

Materials.

Nα-[methyl-3H]histamine ([3H]NAMH) (specific activity 79.7 Ci/mmol), [3H]histamine (specific activity 10.6 Ci/mmol), Microscint-O scintillation liquid, and GF/C filter plates were purchased from PerkinElmer (Groningen, The Netherlands). Clobenpropit-BODIPY-630/650 (clo-BDY, CA200843) was purchased from Hello Bio (Bristol, UK), and BODIPY-FL-histamine (FL-histamine) was purchased from Setareh Biotech (Eugene, OR). Thioperamide and clobenpropit were purchased from Abcam (Cambridge, UK), Trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(1-pyrrolidinylmethyl)phenyl]-cyclobutanecarboxamide 4-methylbenzenesulfonate (PF-03654746) and 4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile (ABT-239) were obtained from Axon Medchem (Groningen, The Netherlands), and S-α-methylhistamine (SAMH) and Nα-methylhistamine (NAMH) were obtained from Tocris (Abingdon, UK). Pitolisant and (4-Cyclopropyl-piperazin-1-yl)-(4-morpholin-4-ylmethyl-phenyl)-methanone bavisant (JNJ31001074) were obtained from Griffin Discoveries (Amsterdam, The Netherlands). A-331440 [(4′-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile] was synthesized at the Department of Radiology and Nuclear Medicine, VU University Medical Center Amsterdam (Amsterdam, The Netherlands) and is commercially available at Tocris. GSK1004723 (4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone) was a kind gift from Dr. Slack from GlaxoSmithKline (Stevenage, UK) (Slack et al., 2011). GSK189254 (6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methylpyridine-3-carboxamide) was purchased from Advanced ChemBlocks Inc. (Burlingame, CA). All other ligands were synthesized in house as previously described (van der Goot et al., 1992; Jansen et al., 1994; Vollinga et al., 1994, 1995; De Esch et al., 1999; Jablonowski et al., 2003; Lim et al., 2006, 2009; Smits et al., 2008). NanoGlo was obtained from Promega (Madison, WI). Fetal bovine serum was obtained from Bodinco (Alkmaar, The Netherlands), and penicillin/streptomycin was purchased from GE Healthcare (Uppsala, Sweden). Hanks’ balanced salt solution (HBSS) was obtained from Gibco (Thermo Fisher Scientific, Waltham, MA). The cAMP-responsive element (CRE)–driven luciferase reporter gene plasmid pTLNC-21CRE was kindly provided by Dr. Born (National Jewish Medical and Research Center, Denver, CO), whereas the BRET-based cAMP biosensor plasmid pcDNA3.1-(L)-His-CAMYEL (cAMP sensor using YFP-Epac-RLuc, #ATCC-MBA-277) was purchased from the American Type Culture Collection (Manassas, VA). All other chemicals were bought from standard commercial resources and were of analytical grade.

Cloning of Nluc-hH3R and Nluc-hH4R Fusion Constructs.

The start codons of the human H3R and H4R [Genbank: AF140538 (Lovenberg et al., 1999) and AY136745, respectively] were substituted with a Kpn2I restriction site sequence using polymerase chain reaction. The cDNA plasmid encoding the membrane signal peptide of the 5HT3A receptor fused to Nluc was a gift from Dr. Hill (University of Nottingham, Nottingham, UK) (Stoddart et al., 2018b). Flanking KpnI and Kpn2I restriction sites were introduced at the 5′ and 3′ end of this signal peptide–Nluc cDNA by polymerase chain reaction, allowing in-frame fusion to the N terminus of hH3R (Lovenberg et al., 1999) and hH4R in the pcDEF3 mammalian expression vector (Goldman et al., 1996). All generated constructs were validated by sequencing (Eurofins Genomics, Ebersberg, Germany).

Cell Culture and Transfection.

Human embryonic kidney 293T cells (HEK293T cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (100 µg/ml), and streptomycin (50 µg/ml) at 37°C with 5% CO2. HEK293T cells were seeded (2 × 106 cells/10 cm2 dish) and transiently transfected the next day with indicated amounts of cDNA using 25 kDa linear polyethyleneimine (PEI). The total amount of cDNA was kept constant at 5 µg per dish by the addition of complementary amounts of pcDEF3 plasmid. DNA/PEI (1:4 ratio) mixtures were preincubated in 150 mM NaCl solution for 20 minutes at 22°C before addition to the cells (Nijmeijer et al., 2013a).

Radioligand-Binding Assays.

HEK293T cells were transfected with 2.5 µg of DNA encoding for wild-type and Nluc-fused hH3R or hH4R. After 48 hours, cells were collected in phosphate-buffered saline (PBS) and centrifuged at 1900g for 10 minutes. The cell pellet was resuspended in 1 ml of PBS per culture dish and centrifuged at 1900g for 10 minutes. Supernatant was discarded and the cell pellet was stored at −20°C until the day of the experiment. Cells were resuspended in binding buffer (50 mM Tris-HCl, pH 7.4, 22°C) and disrupted using a Branson sonifier 250 (Boom bv., Meppel, The Netherlands) on the day of the experiment.

Saturation radioligand binding was performed on 100 µl of cell homogenates expressing the hH3R, Nluc-hH3R, hH4R, or Nluc-hH4R with increasing concentrations of [3H]NAMH or [3H]histamine for 2 hours at 25°C. Nonspecific binding was measured in the presence of 100 µM clobenpropit or 10 µM JNJ7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine). Equilibrium competition binding was performed on 100 µl of cell homogenates expressing the hH3R with ∼2 nM [3H]NAMH or expressing the hH4R with ∼10 nM [3H]histamine in the absence and presence of increasing concentrations of unlabeled ligands for 2 hours at 25°C. Incubations were terminated by rapid filtration over a 0.5% PEI-coated 96-well GF/C filter plate followed by five rapid wash steps with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4, 4°C) using a PerkinElmer 96-well Filtermate-harvester. The GF/C filter plates were dried at 55°C, and 25 µl of Microscint-O scintillation liquid (PerkinElmer, Groningen, The Netherlands) was added per well. Filter-bound radioactivity was measured using a Microbeta Wallac Trilux scintillation counter (PerkinElmer) after a 300-minute delay.

NanoBRET-Based Binding Assays.

HEK293T cells were transfected with cDNA encoding 0.5 µg of Nluc-hH3R or Nluc-hH4R. The next day, cells were transferred to poly-l-lysine–coated black 96-well plates (50,000 cells/well) and grown for an additional 24 hours. Equilibrium saturation binding of fluorescent clo-BDY or FL-histamine to intact cells expressing Nluc-hH3R and Nluc-hH4R was measured in a total volume of 100 µl of HBSS for 2 hours at 25°C. Nonspecific binding was determined by 100 µM clobenpropit or 10 µM JNJ7777120 for Nluc-hH3R and Nluc-hH4R, respectively. To determine the affinity (pKi) of unlabeled ligands for the Nluc-hH3R or Nluc-hH4R, the cells were incubated with 50 nM clo-BDY or 1 µM FL-histamine in combination with increasing concentrations of unlabeled ligands (ranging from 10−5 to 10−11 M) in HBSS for 2 hours at 25°C. Binding of clo-BDY and FL-histamine to the Nluc-hH3R or Nluc-hH4R was detected as a BRET ratio upon the addition of 3.2 µl/ml NanoGlo (Promega, Madison, WI) using the PHERAstar plate reader (BMG Labtech, Ortenberg, Germany) at 475 nm (15-nm bandpass) and >610 nm (longpass) for clo-BDY and 460 nm (80-nm bandpass) and 535 nm (15-nm bandpass) for FL-histamine.

To measure clo-BDY-binding kinetics, Nluc-hH3R–expressing cells were preincubated with 3.2 µl/ml NanoGlo (Promega) in HBSS for 15 minutes. Next, for association assays, 50, 100, or 200 nM clo-BDY was added to the cells and nonspecific binding was measured in the presence of 100 µM clobenpropit. For the competition association assay, 100 nM clo-BDY was added in the absence and presence of unlabeled ligands (10- or 40-fold Ki) in a total volume of 200 µl. In the case of dissociation assays, cells were preincubated with 100 nM clo-BDY for 2 hours before initiation of the dissociation by addition of 10 µM clobenpropit. Clo-BDY binding to the Nluc-hH3R was directly measured every minute for 2 hours using a PHERAstar plate reader (BMG) at 460 nm (80-nm bandpass) and >610 nm (longpass).

CRE-Luciferase Reporter Gene Assay.

HEK293T cells were transiently transfected with 2.5 µg of CRE-luc DNA in combination with 50 ng of Nluc-hH3R or 1000 ng of Nluc-hH4R, hH3R, or hH4R DNA. The next day, cells were transferred to white poly-l-lysine–coated 96-well plates (50,000 cells/well) and grown for an additional 24 hours. The cells were stimulated with increasing ligand concentrations in the absence or presence of a single concentration of antagonist in Dulbecco’s modified Eagle’s medium supplemented with 1 µM forskolin at 37°C and 5% CO2. The incubations were terminated after 6 hours by replacing the medium with 25 µl/well luciferase assay reagent [0.83 mM ATP, 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 µM Na2PO4, 38.9 mM Tris-HCl (pH 7.8), 0.39% glycerol, 0.03% Triton-X-100, and 2.6 µM dithiothreitol]. Luminescence was measured with a Mithras plate reader (Berthold, Bad Wildbad, Germany) after 30 minutes at 37°C.

hH3R Functional Recovery Assay after Antagonist Washout.

HEK293T cells were transfected with 0.5 µg of hH3R and 3 µg of CAMYEL DNA (Scholten et al., 2012). The next day, cells were transferred to a poly-l-lysine–coated white 96-well plate and preincubated overnight with 1 µM hH3R antagonist pitolisant or ABT-239 at 37°C. Next, cells were washed twice with PBS at different time points and allowed to re-equilibrate in HBSS at 37°C. The cells were incubated with 5 µM coelenterazine-h and 40 µM 3-isobutyl-1-methylxanthine for 5 minutes, followed by a stimulation with 1 µM histamine. After 10 minutes, 1 µM forskolin was added and luminescence was measured at 480 nm (20-nm bandpass) (RLuc) and 540 nm (40-nm bandpass) (BRET) using the Mithras multimode reader.

Data Analysis.

Data were analyzed using GraphPad Prism 7.02 (GraphPad Software, San Diego, CA). Data shown are means ± S.D. of at least three independent experiments performed in triplicate, unless stated differently. BRET ratio was calculated by dividing the fluorescent acceptor signal by the signal of the luminescent donor. Competition-binding curves were fitted to a one-sited binding model to quantify the IC50 value, and equilibrium dissociation constants (Ki) of unlabeled ligands were subsequently calculated using the Cheng-Prusoff equation (see equation 1) (Cheng and Prusoff, 1973):

Embedded Image(1)

where [L] is the concentration of labeled ligand, and KD is the equilibrium dissociation constant of the labeled ligand.

Association and dissociation data were fitted and subjected to an extra-sum-of square F-test to compare between one- or two-phase association and one- or two-phase exponential decay models. Competition association curves were fitted to Motulsky and Mahan model (see equation 2) (Motulsky and Mahan, 1984):

Embedded Image(2)

where k1 and k3 (M−1·min−1) are the association rate constants (kon), whereas k2 and k4 (min−1) are the dissociation rate constants (koff) of labeled and unlabeled ligand, respectively; [L] and [I] are the concentrations of labeled and unlabeled ligand, respectively; Bmax is the maximum specific binding sites; and Y is the specific binding.

Kinetic “KD” was calculated according to eq. 3:

Embedded Image(3)

Residence time (RT) indicates how long a ligand stays bound to its receptor (Copeland et al., 2006; Hoffmann et al., 2015; Copeland, 2016) and was calculated using eq. 4:

Embedded Image(4)

The correlation between data sets was analyzed by Deming regression, and the Pearson correlation coefficient (r2) was calculated using GraphPad Prism 7.02.

Results

Pharmacological Characterization of Nluc-Tagged hH3R and hH4R.

To confirm that the N-terminally fused Nluc does not affect hH3R and hH4R function, radioligand saturation binding was measured on HEK293T cell homogenates transiently expressing Nluc-hH3R or Nluc-hH4R and compared with binding to their wild-type counterparts. The radioligand [3H]NAMH displayed similar (P = 0.38, Student’s t test) affinities for the wild-type hH3R (KD = 1.37 ± 0.3 nM, Bmax = 1.5 ± 0.4 pmol/mg, n = 5) and the Nluc-hH3R (KD = 1.78 ± 0.8 nM, Bmax = 11 ± 10 pmol/mg, n = 7), indicating that N-terminal fusion to Nluc does not significantly affect the orthosteric hH3R ligand-binding site (Fig. 1A). Likewise, saturation binding of [3H]histamine to wild-type hH4R and Nluc-hH4R revealed similar (P = 0.22, Student’s t test) KD values of 4.4 ± 0.6 nM (n = 3) and 2.9 ± 1.4 nM (n = 3), respectively, even though Nluc-hH4R expression was lower (Bmax = 5.6 ± 1.4 pmol/mg and 1.3 ± 0.4 pmol/mg for hH4R and Nluc-hH4R, respectively) (Fig. 1B).

Fig. 1.
Fig. 1.

Pharmacological characterization of Nluc-fused hH3R and hH4R. (A) Specific binding of [3H]NAMH to HEK293T cell homogenates expressing wild-type (wt) hH3R or Nluc-hH3R. (B) Specific binding of [3H]histamine to HEK293T cell homogenates expressing wild-type hH4R or Nluc-hH4R. Ligand-induced Nluc-hH3R (C) and Nluc-hH4R (D) activation as measured by CRE-luc reporter gene assay in transiently cotransfected HEK293T cells. Representative graphs of at least three experiments performed in triplicate are shown. RLU, relative luminescence units.

Stimulation of HEK293T cells transiently expressing Nluc-hH3R or Nluc-hH4R with histamine resulted in a concentration-dependent inhibition of 1 µM forskolin-induced CRE reporter gene activity with potency (pEC50) values of 8.6 ± 0.1 and 8.3 ± 0.3, respectively, confirming that N-terminal Nluc fusion to these receptors did not affect their signaling capacity as compared with wild-type hH3R (pEC50 = 8.7 ± 0.1) and hH4R (pEC50 = 8.2 ± 0.2) (data not shown). In contrast, the fluorescent ligand FL-histamine (Supplemental Fig. 1) did not induce Nluc-hH3R or Nluc-hH4R–mediated inhibition of forskolin-induced CRE reporter gene activity. Clobenpropit acted as an antagonist and full agonist at the Nluc-hH3R (α = 0.0) and Nluc-hH4R (pEC50 = 8.8 ± 0.4, α = 1.0 ± 0.1), respectively (Fig. 1, C and D), although previously reported for wild-type hH3R as an inverse agonist and hH4R agonist (Lim et al., 2005). Its fluorescent derivative clo-BDY (structure not disclosed by manufacturer) acted as an antagonist at Nluc-hH3R and full agonist (pEC50 = 8.0 ± 0.1, α = 1.0 ± 0.0) at Nluc-hH4R (Fig. 1, C and D). Indeed, hH3R-mediated inhibition of forskolin-induced CRE reporter gene activity in response to increasing concentrations of histamine was antagonized by 5 µM FL-histamine or 1 µM clo-BDY, resulting in a parallel right shift of the histamine concentration-response curve (Supplemental Fig. 2A). Surprisingly, FL-histamine (10 µM) did not affect hH4R-mediated CRE reporter gene activity in response to increasing concentrations of histamine (Supplemental Fig. 2B), suggesting that FL-histamine does not interact with the hH4R.

NanoBRET-Based Saturation and Competition Binding on Intact Cells Expressing Nluc-hH3R and Nluc-hH4R.

Binding of red fluorescent clo-BDY or green fluorescent FL-histamine to living intact HEK293T cells expressing either Nluc-hH3R or Nluc-hH4R was quantified as NanoBRET between the luminescent Nluc donor and the fluorescent BODIPY acceptor moiety. Incubation of cells expressing either Nluc-hH3R or Nluc-hH4R for 2 hours at 25°C with increasing concentrations of clo-BDY resulted in a saturable increase in NanoBRET signals under equilibrium conditions, resulting in KD values of 13 ± 1.9 and 70 ± 30 nM for Nluc-hH3R and Nluc-hH4R, respectively (Fig. 2, A and C). Saturation binding of FL-histamine displayed moderate affinity KD = 427 ± 87 nM for the Nluc-hH3R (Fig. 2E). Surprisingly, no specific binding could be observed on cells expressing the Nluc-hH4R if tested up to a concentration of 10 μM (data not shown), which is in contrast to the previously reported FL-histamine binding to hH4R on murine bone marrow–derived mast cells with a KD of 2.1 μM as measured by flow cytometry (Mirzahosseini et al., 2015).

Fig. 2.
Fig. 2.

NanoBRET equilibrium saturation binding of clo-BDY on Nluc-hH3R (A), Nluc-hH4R (C), and FL-histamine on Nluc-hH3R (E) transiently expressed on intact HEK293T cells. NanoBRET competition binding of clo-BDY to the Nluc-hH3R (B) and Nluc-hH4R (D), and FL-histamine on Nluc-hH3R (F) transiently expressed on intact HEK293T cells. Representative graphs of at least three experiments performed in triplicate are shown.

Coincubation of a single concentration of clo-BDY (50 nM) or FL-histamine (1 µM) in combination with a selection of unlabeled ligands and cells expressing either Nluc-hH3R or Nluc-hH4R for 2 hours at 25°C resulted in a concentration-dependent decrease in NanoBRET by histamine and clobenpropit, indicating that these unlabeled ligands compete with the fluorescent ligand for binding to both the Nluc-hH3R and Nluc-hH4R (Fig. 2, B, D, and F). In contrast, the hH3R-selective antagonist pitolisant only competed with clo-BDY and FL-histamine for binding to Nluc-hH3R (Fig. 2, B and F) but not for the binding of clo-BDY to Nluc-hH4R (Fig. 2D). This selectivity profile toward H3R was previously observed in radioligand-binding studies on wild-type hH3R and hH4R (Ligneau et al., 2007). Oppositely, the hH4R-selective antagonist JNJ7777120 only displaced clo-BDY from Nluc-hH4R (Fig. 2D) but hardly from Nluc-hH3R (Fig. 2B), which is in line with its previously reported 300-fold lower affinity for the H3R compared with the H4R (Lim et al., 2005).

Binding affinities (pKi) of unlabeled ligands for Nluc-hH3R and Nluc-hH4R were calculated from the NanoBRET-based competition-binding assays using the Cheng-Prusoff equation (Cheng and Prusoff, 1973) and subsequently compared with pKi values that were derived from radioligand competition binding to wild-type hH3R or hH4R (Table 1). A clear difference is observed between agonists and antagonists in the linear correlation of their pKi values for the hH3R obtained in competition with either clo-BDY or [3H]NAMH (Fig. 3A). Agonists consistently displayed lower binding affinities in the NanoBRET-based binding assay as compared with the radioligand-binding assay with a correlation of r2 = 0.8556 (P = 0.003). In contrast, antagonists tended to display more comparable affinities for hH3R and Nluc-hH3R in both assay formats with a correlation of r2 = 0.8008 (P = 0.015). A similar difference in agonist affinities for the hH3R is observed when comparing FL-histamine with [3H]NAMH competition-binding curves. Again, antagonists tended to display more comparable pKi values in both NanoBRET- and radioligand-based assays (Supplemental Table 1). Indeed, pKi values of unlabeled ligands obtained with either “red” tracer clo-BDY or “green” tracer FL-histamine in Nluc-hH3R competition-binding assays showed a high overall linear correlation (r2 = 0.9794, P < 0.0001) (Fig. 3B).

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TABLE 1

Affinity of H3 and H4 ligands on the wild-type hH3R (wt-hH3R) and wt-hH4R using radioligand and Nluc-hH3R and Nluc-hH4R using NanoBRET-based competition binding assays

Data represent the mean ± S.D. of at least three experiments performed in triplicate.

Fig. 3.
Fig. 3.

Correlation plots of equilibrium dissociation constants (pKi) of ligands for Nluc-hH3R (A and B) and Nluc-hH4R (C) as obtained by radioligand or NanoBRET-based competition-binding assays. Correlation of radioligand- and NanoBRET-based pKi values as determined by clo-BDY as tracer (A and C). (B) Correlation between pKi of ligands measured in a NanoBRET-based competition binding assay versus fluorescent clo-BDY or FL-histamine on Nluc-hH3R. Deming linear regression was used for data fitting, and dotted line shows line of unity.

The NanoBRET-based hH4R competition-binding assays on intact cells yielded lower pKi values for all tested ligands as compared with radioligand competition-binding assays on cell homogenates (Table 1). Yet, a correlation was observed for the pKi values of both agonists (r2 0.7673, P = 0.004) and antagonists (r2 0.9184, P = 0.01) between the two hH4R-binding assay formats (Fig. 3C).

Binding Kinetics of Fluorescent Clo-BDY on Nluc-hH3R Measured by NanoBRET.

As both clo-BDY and FL-histamine display similar binding affinities of unlabeled ligands in equilibrium competition binding, clo-BDY was selected as a fluorescent ligand for real-time detection of ligand binding due to its higher affinity and smaller spectral overlap with NanoLuc. Real-time detection of NanoBRET upon the addition of three concentrations (approximately 4×, 8×, and 16× KD) of clo-BDY to intact HEK293T cells expressing Nluc-hH3R at 25°C revealed monophasic association binding of clo-BDY to the Nluc-hH3R with globally fitted kon and koff values of 2.0 ± 1.4 × 105 M−1·min−1 and 0.048 ± 0.02 min−1, respectively (Fig. 4A). The reciprocal of the koff value indicated that clo-BDY has an RT of 23 ± 8.0 minutes on the Nluc-hH3R. Calculation of the equilibrium dissociation constant from the ratio of koff to kon (KD = koff/kon) revealed a 39-fold lower “kinetic” KD value (= 513 ± 272 nM) of clo-BDY for Nluc-hH3R as compared with its KD (= 13 ± 1.9 nM) obtained from the saturation binding curve at equilibrium (Fig. 2A).

Fig. 4.
Fig. 4.

Real-time detection of clo-BDY binding to Nluc-hH3R by NanoBRET. (A) Association of three concentrations of clo-BDY. Competition association assay of 100 nM clo-BDY in competition with clobenpropit (B), histamine (C), pitolisant (D), bavisant (E), and ABT-239 (F) on the Nluc-hH3R transiently expressed on HEK293T cells at 25°C. Representative graphs of at least three experiments performed in triplicate are shown.

Binding Kinetics of Unlabeled Ligands on Nluc-hH3R Measured by NanoBRET.

Real-time measurement of the binding of clo-BDY (100 nM) to the Nluc-hH3R in the absence and presence of unlabeled hH3R ligands allowed the determination of the binding rate constants of the latter by using the Motulsky and Mahan method (Motulsky and Mahan, 1984). A shallow initial overshoot followed by a lower clo-BDY “steady-state” binding to Nluc-hH3R was observed in the presence of 10 nM (40× Ki) unlabeled clobenpropit, which indicates slower dissociation kinetics for unlabeled clobenpropit to the Nluc-hH3R as compared with clo-BDY (Fig. 4B). In contrast, clo-BDY binding to Nluc-hH3R gradually reached equilibrium in the presence of 5011 nM (10× Ki) unlabeled histamine, indicating that this endogenous hH3R agonist has faster receptor-binding kinetics for the Nluc-hH3R in comparison with clo-BDY (Fig. 4C). Indeed, unlabeled clobenpropit and histamine had 1.8-fold lower and 1.9-fold higher koff values, respectively, as compared with clo-BDY (Table 2), resulting in a proportionally longer (44 ± 21 minutes) and shorter (12 ± 3.8 minutes) RT on Nluc-hH3R than clo-BDY. The agonists imetit and NAMH displayed faster binding kinetics as compared with clo-BDY, with RT values of 18 ± 12 and 18 ± 7.4 minutes, respectively (Table 2). The antagonist thioperamide displayed comparably slow binding kinetics as unlabeled clobenpropit, whereas antagonists A-331440 and [4′-{3-([R,R]2,5-dimethyl-pyrrolidin-1-yl)-propoxy}-biphenyl-4-yl]-morpholin-4-yl-methanone (A-349821) bound the Nluc-hH3R with faster kinetics and RTs of 20 ± 14.6 and 17 ± 6.7 minutes, respectively (Table 2). In this assay, the koff value (0.066 ± 0.02 minute−1) of unlabeled A-349821 on intact HEK293T cells was similar to the previously observed dissociation rate constant for the dissociation of prebound [3H]A-349821 from membranes of hH3R-expressing C6 cells (Witte et al., 2006).

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TABLE 2

Kinetic parameters of unlabeled ligands, determined by competition association assays of clo-BDY to Nluc-hH3R

Data represent the mean ± S.D. of at least three experiments performed in triplicate.

The only currently marketed hH3R-targeting antagonist, pitolisant (Wakix), bound the Nluc-hH3R with relatively fast kinetics (kon = 1.4 ± 1.0 × 106 M−1·min−1 and koff = 0.086 ± 0.07 minute−1), resulting in an RT of 17 ± 10 minutes (Fig. 4D; Table 3). Interestingly, considerable variation was observed in the target residence time of five hH3R antagonists that have been or are currently in clinical trials. The RT of bavisant (Fig. 4E) and PF-03654746 was in the same range as pitolisant, with RT values of 8.4 ± 2.3 and 15 ± 4.1 minutes, respectively (Table 3). In contrast, considerably longer residence times for binding to Nluc-hH3R were observed for GSK1004723 (59 ± 83 minutes) and ABT-239 (75 ± 52 minutes), resulting in an initial overshoot of clo-BDY binding to Nluc-hH3R (Fig. 4F).

View this table:
TABLE 3

Kinetic parameters of H3R drug candidates, determined by competition association assay with clo-BDY on the Nluc-hH3R

Data represent the mean ± S.D. of at least three experiments performed in triplicate.

The “kinetic pKD” calculated from the observed kon and koff values of the unlabeled ligands in the competitive association-binding assay revealed a linear relationship (r2 = 0.7398, P = 0.0007) with the corresponding pKi values that were derived from competition-binding curves under equilibrium conditions using the same NanoBRET technology (Fig. 5A; Tables 2 and 3). However, the calculated kinetic pKD values were up to 50-fold lower than the observed pKi (Fig. 5A). Interestingly, ligand affinity (pKi) of unlabeled ligands was linearly correlated (r2 = 0.7789, P = 0.0003) to the log kon (Fig. 5B), but no correlation (r2 = 0.067, P = 0.441) was found between pKi and log koff values (Fig. 5C), suggesting that the on-rate of ligands drives hH3R affinity (Sykes et al., 2009; Guo et al., 2016). Plotting the logarithm of kon versus the koff values illustrates that ligands with the same affinity, as indicated by the diagonal lines, can have considerably different kinetics (Fig. 5D).

Fig. 5.
Fig. 5.

Comparison of the affinity (pKi) of unlabeled ligands for the Nluc-hH3R obtained from equilibrium NanoBRET assays with their association and dissociation rate constants and kinetic “pKD” from real-time measurements (Table 2). Deming linear regression was used for data fitting of pKi versus pKD (A), pKi versus log kon (B), and pKi versus log koff (C). Distribution of log kon versus log koff in relation to their kinetic “KD” as represented by diagonal lines (D). Dotted line shows line of unity.

Duration of Functional hH3R Antagonism.

Next, the duration of H3R antagonism was functionally measured upon induction of prebound antagonist dissociation by washout of unbound ligand to evaluate the impact of the RT (= 1/koff) derived from the NanoBRET competitive association assay. To this end, the recovery rate of hH3R responsiveness to histamine (1 µM) stimulation was measured in transiently transfected HEK293T cells by using the cAMP-sensitive BRET-based biosensor CAMYEL at 37°C. Overnight preincubation with 1 µM hH3R antagonists pitolisant or ABT-239 (pKi values were 8.6 ± 0.0 and 9.0 ± 0.2, respectively, in NanoBRET-based binding on intact cells) followed by a washout of unbound antagonist resulted in a full recovery of hH3R responsiveness for the short-RT (17 minutes) antagonist pitolisant within 1.5 hours. In contrast, the prebound long-RT (75 minutes) antagonist ABT-239 showed a much-slower recovery of hH3R responsiveness to histamine stimulation, and 60% of hH3R-mediated signaling was still blocked 4.5 hours after its dissociation was initiated (Fig. 6).

Fig. 6.
Fig. 6.

Functional washout of 1 µM pitolisant or ABT-239 from the hH3R and their ability to antagonize histamine-induced hH3R activation. Cells were incubated overnight with either 1 µM pitolisant or ABT-239. Subsequently, cells were washed and allowed to re-equilibrate for different time periods before stimulation with histamine and detection of cAMP level using CAMYEL biosensor. Representative graph of three experiments performed in duplicate is shown.

Discussion

The availability of fluorescent ligands in combination with the recently engineered Nluc luciferase allows the development of NanoBRET-based ligand-receptor-binding assays (Stoddart et al., 2015, 2018a,b; Soave et al., 2016). Since NanoBRET only occurs if the fluorescent ligand is bound to the Nluc-tagged GPCR, this assay can be performed in a homogeneous format without additional washing steps to separate unbound from bound fluorescent ligand and is, therefore, well suited to detect real-time ligand binding to GPCRs that are expressed on living cells. Quantification of binding parameters of a ligand for its GPCR on living cells is functionally more relevant as compared with measurements on membrane preparations or cell homogenates, as used buffer and intracellular interacting proteins can affect the latter assays (Emami-Nemini et al., 2013; Vauquelin et al., 2015; Bosma et al., 2017; Vanderheyden and Benachour, 2017; Stoddart et al., 2018). Here, a NanoBRET-based binding assay for the Nluc-hH3R and Nluc-hH4R on intact cells was developed utilizing the commercially available fluorescent ligands clo-BDY and FL-histamine.

As shown in this study, unlabeled agonists displayed higher affinity for the hH3R in cell homogenates as determined by [3H]NAMH displacement in comparison with the NanoBRET-based binding assay on intact cells. Indeed, higher-affinity agonist-bound GPCR ternary complexes can be formed in membrane preparations or cell homogenates with their associated G protein in the absence of free guanine nucleotides, as compared with the more-transient ternary complexes in intact cells resulting in lower agonist-binding affinities (Witte et al., 2006; Strange, 2008; Chabre et al., 2009; Emami-Nemini et al., 2013). Interestingly, a similar difference in the affinity of unlabeled agonists for the hH3R was observed in competition-binding experiments on cell homogenates using agonist [3H]NAMH versus antagonist [125I]iodophenpropit as radioligands (Bongers et al., 2007). Comparison of pKi values of unlabeled ligands derived from the NanoBRET-based competition binding using the fluorescent antagonist clo-BDY with those obtained from [125I]iodophenpropit displacement revealed similar binding affinities in both assay formats (r2 0.9239, P < 0.0001) without an obvious discrepancy between unlabeled H3R agonists and H3R antagonists (Fig. 7). In contrast to the [3H]NAMH-binding assay, both NanoBRET-based clo-BDY and [125I]iodophenpropit competition binding assays are performed in buffers containing near-physiologic (∼140 mM) NaCl concentrations (Bongers et al., 2007). Allosteric binding of sodium to GPCRs often reduces agonist affinity by stabilizing inactive receptor conformations but may also differently affect the binding of antagonists to the orthosteric binding site (Clark and Hill, 1995; Schnell and Seifert, 2010; Katritch et al., 2014; Wittmann et al., 2014; Newton et al., 2016; Hishinuma et al., 2017; Schiffmann and Gimpl, 2018). Nonetheless, most antagonists displayed affinities in the NanoBRET assay that were comparable to the radioligand-based competition-binding assay. Lower hH4R-binding affinities for both agonist and antagonists were also observed in the NanoBRET-based binding assay on intact cells as compared with radioligand binding to cell homogenates, despite the fact that both labeled clo-BDY and [3H]histamine are agonists on the hH4R. Similar to the hH3R, the presence of allosteric sodium in the NanoBRET buffer may reduce affinities of both agonists and antagonists for the hH4R. In addition, competitive binding experiments and the Cheng-Prusoff equation assume reversible interaction between competing ligands and receptor. However, bound fluorescent agonists may induce receptor internalization during the course of a binding experiment on intact cells, which may result in the observation of lower pKi values for unlabeled ligands as compared with binding assays on cell homogenates (Cheng and Prusoff, 1973; Guo et al., 2010).

Fig. 7.
Fig. 7.

Comparison of pKi of unlabeled ligands for the hH3R obtained from equilibrium NanoBRET assays with affinities obtained with [125I]iodophenpropit from Bongers et al. (2007). Deming linear regression was used for data fitting. Dotted line shows line of unity.

The NanoBRET-based binding assay can detect binding of a fluorescent ligand to living cells expressing the Nluc-tagged receptor of interest with a high resolution in time, which also allows for the measurement of a full association-binding curve from a single sample (Hansen et al., 2017; Stoddart et al., 2018). Detection of the association binding of a fluorescent probe in the presence of unlabeled ligands allows quantification of the kinetic rate constants and KD (= koff/kon) values of the latter. Since binding rate constants are sensitive to temperature, the ionic composition of buffer, and can be different in living cells versus cell homogenates or membrane preparations (Sykes et al., 2010; Vanderheyden and Benachour, 2017), one might anticipate that absolute rate constants of the few ligands that were previously reported in radioligand-binding studies might be different from those obtained using the current NanoBRET binding assay on living cells. Indeed, the hH3R antagonist GSK1004723 displayed 5-fold faster dissociation kinetics in the NanoBRET binding assay compared to that previously reported (Slack et al., 2011), despite the lower temperature (25°C instead of 37°C) used in NanoBRET. In contrast, association and dissociation binding of the antagonist [3H]A-349821 to membranes expressing the hH3R revealed a 220-fold higher kon value and similar koff value as compared with the NanoBRET-based competition association assay (Witte et al., 2006).

Although a linear correlation was observed between pKi and kinetic pKD values of unlabeled hH3R ligands in the NanoBRET-based binding assays, the kinetic pKD values were consistently lower than the affinities that were obtained from the equilibrium binding assay. Hence, the absolute values of the determined binding rate constants in this competitive association binding should not be overinterpreted. However, evaluation of the functional recovery rate of hH3R responsiveness to histamine stimulation upon dissociation of prebound long- and short-RT antagonists, under assay conditions comparable to the NanoBRET-based binding assay, revealed that obtained RT values are indeed very indicative of the duration of hH3R antagonism of these antagonists.

Hitherto, only pitolisant (Wakix) is approved as an hH3R-targeting antagonist to increase wakefulness and alertness in patients suffering from narcolepsy. Pitolisant was found to have a moderate RT (17 minutes) on the hH3R, which consequently resulted in a relatively fast target recovery rate (Fig. 6). From the hH3R antagonists that have been or are currently in clinical trials, bavisant and PF-03654746 have an RT comparable to pitolisant, whereas ABT-239 and GSK1004723 show substantially longer RTs. Recently, a new trial was initiated with bavisant to assess excessive daytime sleepiness in Parkinson’s disease patients (NCT03194217). The long-residence-time antagonist GSK1004723 displayed a lack of in vivo efficacy, whereas ABT-239 failed to enter clinical trials due to drug safety issues (Hancock, 2006; Daley-Yates et al., 2012). Insomnia is frequently reported as a side effect of hH3R antagonists in clinical trials, and hence long-RT ligands might further enhance this adverse event (Wijtmans et al., 2007; Brioni and Esbenshade, 2011; Sadek et al., 2016; Schwartzbach et al., 2017). Moreover, it was proposed that different ligands can address distinct hH3R-related disorders (Esbenshade et al., 2006; Wijtmans et al., 2007; Sander et al., 2008). Therefore, it remains to be seen what influence ligand kinetics have on the plethora of hH3R-related therapeutic indications.

In conclusion, the NanoBRET-based competition equilibrium binding assay is a valuable tool to determine the binding parameters of unlabeled ligands for their receptors on living cells at reasonably high throughput under experimental conditions that better correspond to those used in functional assays, as compared with the traditional radioligand-binding studies on cell homogenates or isolated membranes.

Acknowledgments

Mouad El Mandili, Laura Bothof, and Rabia Asik are acknowledged for their help with Nluc-hH3R and Nluc-hH4R experiments, and Cas van der Horst is acknowledged for his help in radioligand displacement assays.

Authorship Contributions

Participated in research design: Mocking, Vischer, Leurs.

Conducted experiments: Mocking, Verweij.

Performed data analysis: Mocking, Verweij.

Wrote or contributed to the writing of the manuscript: Mocking, Vischer, Leurs.

Footnotes

    • Received June 13, 2018.
    • Accepted September 19, 2018.

  • This work was supported by The Netherlands Organization for Scientific Research (NWO) TOPPUNT [“7 ways to 7TMR modulation (7-to-7)”] [Grant 718.014.002] and ECHO [Grant 711.013.014].

  • https://doi.org/10.1124/mol.118.113373.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

A-331440
(4′-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile
A-349821
([4′-{3-([R,R]2,5-dimethyl-pyrrolidin-1-yl)-propoxy}-biphenyl-4-yl]-morpholin-4-yl-methanone
ABT-239
4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile
BRET
bioluminescence resonance energy transfer
CAMYEL
cAMP sensor using YFP-Epac-RLuc
clo-BDY
clobenpropit-BODIPY
CRE
cAMP-responsive element
FL-histamine
BODIPY-FL-histamine
GPCR
G protein–coupled receptor
GSK1004723
4-[(4-Chlorophenyl)methyl]-2-({(2R)-1-[4-(4-{[3-(hexahydro-1H-azepin-1-yl)propyl]oxy}phenyl)butyl]-2-pyrrolidinyl}methyl)-1(2H)-phthalazinone
GSK189254
6-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methylpyridine-3-carboxamide
HBSS
Hanks’ balanced salt solution
HEK293T
human embryonic kidney 293T
hH3R
human H3R
hH4R
human H4R
H3R
histamine H3 receptor, H4R, histamine H4 receptor
JNJ31001074
(4-Cyclopropyl-piperazin-1-yl)-(4-morpholin-4-ylmethyl-phenyl)-methanone, A-331440: (4′-[3-(3(R)-(dimethylamino)-pyrrolidin-1-yl)-propoxy]-biphenyl-4-carbonitrile
JNJ7777120
1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine
NAMH
Nα-methylhistamine
[3H]NAMH
Nα-[methyl-3H]histamine [3H]
Nluc
NanoLuc luciferase
PBS
phosphate-buffered saline
PEI
polyethyleneimine
PF-03654746
Trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(1-pyrrolidinylmethyl)phenyl]-cyclobutanecarboxamide 4-methylbenzenesulfonate
RT
residence time
SAMH
s-α-methylhistamine

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Real-Time NanoBRET Binding Assays for the H3R and H4R on Living Cells

Tamara A. M. Mocking, Eléonore W. E. Verweij, Henry F. Vischer and Rob Leurs
Molecular Pharmacology December 1, 2018, 94 (6) 1371-1381; DOI: https://doi.org/10.1124/mol.118.113373
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